COMPOSITIONS AND METHODS FOR SELECTION OF NUCLEIC ACIDS CONTAINING MODIFIED BASES

• US 20140179564A1
• Filed: 10/31/2013
• Published: 06/26/2014
• Est. Priority Date: 11/01/2012
• Status: Abandoned Application

• Current Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Original Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Inventors: Korlach, Jonas, Clark, Tyson A., Turner, Stephen
• Application Number:
  US14/068,293
• Publication Number:
  US 20140179564A1
• Time in Patent Office: Days
• US Class Current: 506/26
• CPC Class Codes: C12N 15/101 by chromatography, e.g. electrophoresis, ion-exchange, reverse phase
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 2525/117 incorporating modified base
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing

Abstract

Methods are provided for reducing the complexity of a population of nucleic acids prior to performing an analysis of the nucleic acids, e.g., sequence analysis. The methods result in a subset of the initial population enriched for a desired property, or lacking nucleic acids having an undesired property. The methods are particularly useful for analyzing populations having a high degree of complexity, e.g., chromosomal-derived DNA, whole genomic DNA, or mRNA populations. In addition, such methods allow for analysis of pooled samples.

Claims

1. A method of generating a pool of sequencing templates enriched for a type of modified base, the method comprising:

a) fragmenting a nucleic acid sample comprising the type of modified base, thereby generating a mixture comprising a first subset of nucleic acid fragments comprising the type of modified base and a second subset of nucleic acid fragments that do not comprise the type of modified base;

b) linking adapters to nucleic acid fragments in both the first subset and the second subset; and

c) retaining the nucleic acid fragments in the first subset from the mixture, thereby generating a pool of sequencing templates enriched for the type of modified base.

2. The method of claim 1, wherein the retaining comprises binding the type of modified base to an agent linked to an affinity tag.

3. The method of claim 1, wherein the type of modified base is a methylated cytosine.

4. The method of claim 1, wherein the type of modified base is a hydroxymethylated cytosine.

5. The method of claim 1, wherein the adapters are hairpin or stem-loop adapters that link 3′

and 5′

termini at each end of the nucleic acid fragments.

6. The method of claim 5, wherein the adapters added to a first end of the nucleic acid fragments in the first subset are different from the adapters added to a second end of the nucleic acid fragments in the first subset.

7. The method of claim 1, wherein the retaining comprises affinity purifying the nucleic acid fragments.

8. The method of claim 1, wherein said retaining comprises binding driver nucleic acids to the first subset of nucleic acid fragments comprising the type of modified base.

9. The method of claim 8, wherein the driver nucleic acids are complementary to a sequence motif that comprises the modified base in the first subset of nucleic acid fragments.

10. The method of claim 8, wherein the driver nucleic acids are generated from an aliquot of the nucleic acid sample.

11. The method of claim 10, wherein the aliquot is subjected to cleavage with a methyl-dependent restriction endonuclease.

12. The method of claim 11, further comprising subjecting the aliquot to a size selection following the cleavage and prior to the binding.

13. The method of claim 11, further comprising subjecting the aliquot to an amplification reaction following the cleavage and prior to the binding.

14. The method of claim 11, wherein the methyl-dependent restriction endonuclease is MspJI.

15. The method of claim 8, wherein the driver nucleic acids comprise an affinity tag.

16. The method of claim 8, wherein the driver nucleic acids comprise a sequence motif suspected of comprising the type of modified base in the first subset of nucleic acid fragments.

17. The method of claim 8, wherein the binding of the driver nucleic acids to the first subset of nucleic acid fragments is performed in the presence of a strand-exchange protein.

18. The method of claim 1, wherein the nucleic acid fragments in both the first subset and the second subset are not amplified prior to the retaining.

19. The method of claim 1, further comprising removing second subset of nucleic acid fragments that do not comprise the type of modified base.

20. The method of claim 19, wherein the removing comprises nuclease digestion.